Photoactive nanocoating for controlling microbial proliferation on polymeric surfaces

Progress in Organic Coatings 62 (2008) 40–48

Photoactive nanocoating for controlling microbial proliferation on polymeric surfaces Alex Bilyk a , Sheng Li a , James Murphy b , Steven Petinakis a , Katherine Zerdin b , Andrew Scully a,∗ a

CSIRO Materials Science and Engineering, Gate 5 Normanby Road, Clayton, Vic. 3169, Australia b Food Science Australia, 11 Julius Avenue, North Ryde, NSW 2113, Australia Received 22 June 2007; received in revised form 14 August 2007; accepted 13 September 2007

Abstract A novel antimicrobial polymeric surface coating involving photo-activated generation of the well-known microbiocidal agent hydrogen peroxide was investigated. Modified poly(ethylene imine) (PEI) polymers were prepared containing anthraquinone (AQ) moieties attached covalently to the amino functional groups of the PEI chains. These AQ-modified PEI polymers were applied from solution to the surface of corona-treated low-density polyethylene (LDPE) films. Photoreduction of the PEI-bound anthraquinone derivatives occurred on exposure to low energy UV light, resulting in efficient production of hydrogen peroxide from the coated film on exposure to air. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. Keywords: Antimicrobial; Nanocoating; Polyolefin; Corona; Photoreduction

1. Introduction An extensive range of polymeric materials incorporating antimicrobial agents has been investigated for minimising the proliferation of microbes on the surface of food packaging [1–3] and on other polymeric surfaces [4]. In many of the antimicrobial polymers reported to date, the microbial control agent is distributed into the bulk of the material by dissolution or dispersion. Apart from being technically facile, the advantage of this blending approach is that the unbound antimicrobial agent can continuously diffuse to the polymer surface to react with microbes in contact with the surface. Although volatility of the microbial control agent can be beneficial, such as in some food package applications, a substantial amount of the unbound agent can be lost from the polymer due to volatilisation either during melt compounding, or during storage prior to end use. It is, therefore, often desirable that the microbial control agent be immobilized through chemical binding to the

∗ Corresponding author at: CSIRO Materials Science and Engineering, Private Bag 33, Clayton South MDC, Vic. 3169, Australia. Tel.: +61 3 9545 2965; fax: +61 3 9544 1128. E-mail address: [email protected] (A. Scully).

polymer, preferably at the polymer surface in order to maximize the antimicrobial efficacy of the material. Immobilization of antimicrobial agents specifically on the surface of many types of polymeric materials is difficult due to the lack of suitable reactive sites on the surface to enable chemical bonding. Common methods used to functionalise polymeric surfaces include corona discharge, flame treatment, plasma treatment, UV irradiation, electron beam irradiation, chemical oxidation, and surface chemical grafting. Examples of immobilization of antimicrobial agents through covalent attachment to pre-activated surfaces include the grafting of a synthetic bacteriocin to the surface of polystyrene beads pre-activated with poly(ethylene glycol) [5], use of cross-linking agents such as gluteraldehyde and carbodiimides to attach chitosan [6] or glucose oxidase [7] to the surface of plasma-treated bi-oriented polypropylene films or the fungicide benomyl to the surface of Surlyn® film [8], attachment of chitosan to surfaces of electron beam-treated HDPE tubes primed with acrylic acid [9], and the grafting of porphyrin-based photosensitizers via an ethylene diamine bridge onto the surfaces of nylon-6,6 films [10] and fibers [11] after pre-activation of the nylon-6,6 surface with a poly(acrylic acid) scaffold. The antimicrobial effectiveness of hydrogen peroxide is well documented, and dips and sprays comprising hydrogen

0300-9440/$ – see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.porgcoat.2007.09.011

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peroxide are commonly used by food manufacturers to disinfect the surfaces of packaging materials for aseptically packaged foods [12]. Although it is an effective sterilising agent, the handling of hydrogen peroxide solutions in food manufacturing plants can be hazardous, and unwanted oxidation and/or tainting of packaged foods can occur as a result of hydrogen peroxide residues migrating into the food. A material capable of being activated to produce hydrogen peroxide at its surface would avoid the need to use hydrogen peroxide solutions in the food manufacturing environment. Anthraquinone (AQ) compounds are known to readily undergo photoreduction to form the corresponding 9,10dihydoxyanthracene on exposure to UV light in the presence of suitable reductants such as amines or alcohols, and on exposure to oxygen, the 9,10-dihydoxyanthracene compounds rapidly re-oxidize to re-form the original AQ compound with the accompanying formation of hydrogen peroxide [13]. Since the photoreduction of AQ compounds in polymeric materials has been reported [14,15], it might be expected that if an AQ compound, together with a suitable reductant, can be bound to the surface of a polymeric material, then it may be possible to create a material capable of producing hydrogen peroxide at its surface, on exposure to low-power UV light. We have reported previously [16] that application of a polyamine, such as poly(ethylene imine) (PEI), to the surface of polymeric materials after surface treatment using corona or flame can result in greater stability of the functionalised polymer surface, and amines are known [13,17] to be capable of acting as reductants in the photoreduction of AQ compounds. In the present work, we investigated the coating of a nano-scale layer of PEI modified with covalently grafted AQ moieties onto a corona-activated polyethylene film, and the potential of this coating to produce hydrogen peroxide.

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2.1. NMR analysis One- and two-dimensional multi-nuclear spectra were performed using a 500 MHz spectrometer (Bruker DRX) at 500 MHz and 125 MHz for 1 H and 13 C nuclei, respectively, and/or a 200 MHz NMR spectrometer (Bruker AMX or AV) at 200 and 60 MHz for 1 H and 13 C nuclei, respectively. Samples were contained in 5 mm Wilmad NMR tubes. The centre peak of the splitting pattern of residual chloroform at 7.26 ppm (singlet) and 77.16 ppm (triplet), or alternatively, where stated, residual dimethyl sulfoxide at 2.50 ppm and 39.52 ppm were used as references for 1 H and 13 C spectra, respectively. Heteronuclear single quantum correlation (HSQC), heteronuclear multi-bond correlation (HMBC) and correlated spectroscopy (COSY) spectra were used to confirm the structures of the compounds (1) and (2) (the spectra and correlation data are available as supplementary information).

2. Experimental All reagents were obtained from commercial sources, with the exception of anthraquinone-2-sulfonyl chloride which was prepared according to a literature method [18] from anthraquinone-2-sulfonate sodium salt monohydrate. All reagents were used as supplied from the following sources: from Sigma–Aldrich, anthraquinone-2sulfonate sodium salt monohydrate (97%), N-butylamine (99.5%), chloroform-d (99.8 atom% D), benzyltriethylammonium bromide (99%), dimethylsulfoxide-d6 (99.9 atom% D), and epichlorohydrin (99%); from BDH, chloroform (AnalaR), ethyl acetate (AnalaR), magnesium sulfate dried (GPR), methanol (AnalaR), 2-N-methyl2pyrrolidone (GPR), sodium chloride (AnalaR), sodium hydroxide (AnalaR); from Ajax Fine Chemicals, methoxyethanol (A.R), petroleum spirit 60–80 ◦ C (A.R), thionyl chloride (L.R); from Ausprep, dimethylformamide (peptide synthesis grade); from BASF Australia, PEI (WF Lupasol, MW ∼25,000). TLC analysis was performed using Kieselgel 60F254 TLC plates (Merck) and a mineralight® lamp. All reaction solutions were refrigerated upon completion of the reactions, and were filtered through a 0.45 ␮m syringe filter prior to further use.

Quantitative 13 C NMR analysis (Q-13 C) of the modified PEI polymers was performed using chloroform-d as the solvent. The numbering systems used to assist assignment of NMR chemical shifts associated with the anthraquinone compounds synthesized in this work are depicted in structures (1) and (2).

2.2. Synthesis of 2-N-butylaminosulfonyl anthraquinone 2-Sulfonyl chloride anthraquinone (45 g and 150 mol) was added as a solid to a solution of N-butylamine (75 ml and 760 mmol) in methoxyethanol (160 ml) at ambient temperature. The partial suspension was stirred vigorously for 3 h prior to the isolation of the resulting precipitate, which was liberally washed with methanol. The crude product was re-crystallised from methoxyethanol and dried in vacuo prior to further use. Yield 33.4 g (97 mmol and 64.7%); mp 162.9–163.4 ◦ C; 1 H NMR (200 MHz DMSO-d6 ) (the numbering system used is based on that shown in structure (1)): δ 8.44 (1H, d, AQ-1, J = 1.43 Hz), 8.28 (1H, d, AQ-4, J = 8.03 Hz), 8.17–8.22 (1H, dd, AQ-3, J = 1.91 and 8.16 Hz), 8.00–8.10 (2H, m, AQ-5,8),

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7.79–7.87 (2H, m, AQ-6,7), 2.81 (2H, m, 1 ), 1.11–1.44 (4H, m, 2 and 3 ) and 0.77 (3H, t, 4 , J = 7.01 Hz) ppm. 13 C NMR (60 MHz CDCl3 ): 181.82 (AQ-10), 181.78 (AQ-9), 146.14 (AQ2), 135.42 (AQ-9a), 135.08 (AQ-6,7), 133.96 (AQ-4a), 133.19 (AQ-8a,10a), 131.93 (AQ-3), 128.47 (AQ-4), 127.19 (AQ-5,8), 124.95 (AQ-1), 42.68 (1 ), 31.55 (2 ), 19.62 (3 ) and 13.87 (4 ) ppm. 2.3. Synthesis of 2-N-butyl-N-glycidylaminosulfonyl anthraquinone “GLYAQ” (1) A suspension of 2-N-butylaminosulfonyl anthraquinone (20 g and 60 mmol), epichlorohydrin (27 ml and 600 mmol) and tetrabutylammonium bromide (1.87 g and 5.8 mmol) in Nmethylpyrrolidone (60 ml) was held at 80 ◦ C for approximately 2 h. A 50% w/v aqueous sodium hydroxide solution (14 ml and 88 mmol) was then added drop-wise to the resulting mixture. The reaction was cooled to 50 ◦ C after the addition of the base, and was held at this temperature overnight. The solution was filtered prior to cooling, and the retained filtrate was diluted in warm methanol (420 ml). The resulting precipitate formed upon cooling was isolated, washed with chilled methanol, and then dried in vacuo. The isolated product was used without further purification, however, it can be re-crystallised from a chloroform/methanol solvent mixture. Yield 12.1 g (30.3 mmol, 51%); mp 121.8–122.9 ◦ C; 1 H NMR (200 MHz CDCl3 ): δ 8.72 (1H, d, AQ-1, J = 1.89 Hz), 8.46 (1H, d, AQ-4, J = 8.16 Hz), 8.30–8.40 (2H, m, AQ-5,8), 8.24 (1H, dd, AQ-3, J = 1.91 and 8.16 Hz), 7.82–7.90 (2H, m, AQ-6,7), 3.73–3.81(1H, dd, 1 , J = 2.25 and 14.32 Hz), 3.17–3.46 (2H, m, 1 ), 2.95–3.13 (2H, m, 1 and 2 ), 2.77–2.82 (1H, t, 3 , J = 4.16 Hz), 2.54–2.57 (1H, dd, 3 , J = 2.44 and 4.75 Hz), 1.53–1.68 (2H, m, 2 ), 1.24–1.42 (2H, sextet, 3 , J = 7.11 Hz) and 0.92 (3H, t, 4 , J = 7.20 Hz) ppm. 13 C NMR (125 MHz CDCl ): 181.93 (AQ-10), 181.71 (AQ-9), 3 145.52 (AQ-2), 135.71 (AQ-9a), 134.74 (AQ-6,7), 134.15 (AQ-4a), 133.28, 133.25 (AQ-8a,10a), 131.84 (AQ-3), 128.54 (AQ-4), 127.61 (AQ-5,8), 125.87 (AQ-1), 50.78 (1 ), 50.68 (2 ), 49.14 (1 ), 45.21(3 ), 30.49 (2 ), 19.8 (3 ) and 13.6 (4 ) ppm. 2.4. Synthesis of 2-(N-butyl-N-(2-hydroxy-3butylaminopropyl)-aminosulfonyl)-anthraquinone (2) To assist in the characterisation of the polymers prepared in this work, the monomeric amine ring-opened epoxy anthraquinone (2) was synthesised as a model compound. A solution of GLYAQ (500 mg and 1.25 mmol) and nbutylamine (617 ␮l and 6.35 mmol) in methoxyethanol (2 ml) was stirred vigorously under nitrogen at 50 ◦ C. After an overnight period under the stated conditions, analysis by TLC (eluant: 10%MeOH/CHCl3 ) indicated a single product was formed with negligible residual unreacted GLYAQ. The solution was diluted with chloroform and washed with water and brine successively prior to drying over magnesium sulfate. The solvent was removed in vacuo to yield a crude yellow solid. The model compound was isolated by flash chromatography as a yellow crystalline product, eluant: 10%methanol/chloroform:

yield 451 mg (0.95 mmol and 76%). mp 131.4–132.4 ◦ C; 1 H NMR (500 MHz CDCl3 ): δ 8.72 (1H, d, AQ-1, J = 1.89 Hz), 8.45 (1H, d, AQ-4, J = 8.15 Hz), 8.36–8.33 (2H, m, AQ-5,8), 8.22 (1H, dd, AQ-3, J = 1.89 and 8.14 Hz), 7.84–7.88 (2H, m, AQ-6,7), 3.84–3.89(1H, m, 2 ), 3.17–3.36 (4H, m, 1 and 1 overlap), 2.78–2.82 (1H, dd, 3 , J = 3.77 and 12.26 Hz), 2.63–2.69 (3H*, m, 3 and 1 ), 1.46–1.62 (4H, m, 2 and 2 overlap), 1.24–1.38 (4H, m, 3 and 3 overlap), 0.87–0.923 (6H, t(2), 4 and 4 overlap, J = 7.33 Hz) ppm. 13 C NMR (125 MHz CDCl3 ): 181.97 (AQ-10), 181.72 (AQ-9), 145.17 (AQ-2), 135.66 (AQ9a), 134.73, 134.72 (AQ-6,7), 134.06 (AQ-4a), 133.28, 133.24 (AQ-8a,10a), 132.00 (AQ-3), 128.47 (AQ-4), 127.60, 127.57 (AQ-5,8), 126.00 (AQ-1), 67.84 (2 ), 52.18 (1 ), 52.06 (3 ), 49.77 (1 ), 49.45 (1 ), 31.38 (2 ), 30.55 (2 ), 20.25 (3 ), 19.90 (3 ), 13.9 (4 ) and 13.68 (4 ) ppm. *Attributed to the sum of the signal associated with one of the chemically inequivalent protons coupled to C-3 , and the two protons coupled to C-1 that are assumed to be chemically equivalent. If, in fact, the C-1 protons are not chemically equivalent due to residual influence from the stereogenic centre at C-2 , then the difference in their chemical shifts must be beyond the resolution of the NMR instrument used in this work. 2.5. Preparation of PEI modified with GLYAQ PEI-g4.9-GLYAQ: a suspension of GLYAQ (0.109 g and 0.273 mmol) and WF Lupasol (1.039 g) in methoxyethanol (9 ml) was vigorously stirred under nitrogen at 50 ◦ C. After 5 h under these conditions, negligible unreacted GLYAQ could be detected by TLC (eluant: 40%EtOAc/ Pet. Spirit). Elevation of the reaction temperature was found to result in a darkening of the reaction mixture. A representative reaction aliquot was taken on completion of the reaction, and the solvent was removed in vacuo to furnish viscous amber oil. 13 C NMR (125 MHz CDCl3 ): 181.83, 181.64(AQ-9,10), 163.67(CO), 161.89 (CO), 157.84 (CO), 145.53 (AQ-2), 135.48 (AQ-9a), 134.62 (AQ-6,7), 133.9 (AQ-4a), 133.14 (AQ-8a,10a), 131.90 (AQ-3), 128.35 (AQ-4), 127.45 (AQ-5,8), 125.84 (AQ-1), 70.82 (2 ) 65.82 (2 ), 57.52–39.80* (PEI, 1 , 1 and 3 ), 30.24 (2 ), 19.81 (3 ) and 13.65 (4 ) ppm. *Broad band of overlapping signals. PEI-g8.7-GLYAQ: same method as for PEI-g4.9-GLYAQ; GLYAQ (0.256 g and 0.64 mmol), WF Lupasol (1.27 g), methoxyethanol (11 ml). Q-13 C NMR (125 MHz CDCl3 ) (theoretical values shown in parentheses next to the observed integrals): 181.83, 181.60 (2C (2C), AQ-9,10), 145.54 (1C (1C), AQ-2), 135.40 (1.04C (1C), AQ-9a), 134.58 (1.80C (2C), AQ-6,7), 133.87 (1.27C (1C), AQ-4a), 133.14 (1.95C (2), AQ8a,10a), 131.89 (0.96C (1C), AQ-3), 128.26 (0.88C (1C), AQ-4), 127.43 (1.75C (2C), AQ-5,8), 125.85 (0.88C (1C), AQ-1), 70.85 (0.79C (?C)*, 2 ) 68.44 (0.96C (?C)*, 2 ), 57.52–39.80 (102.39C (∼101C), PEI, 1 , 1 and 3 ), 30.24 (0.85C (1C), 2 ), 19.81 (0.98C (1C), 3 ) and 13.65 (0.90C (1C), 4 ) ppm. *The total sum of the theoretical integrals at these shifts equals 1, but the measured integrals of these shifts are affected by the signal from residual chloroform. The reaction products obtained from the above syntheses did not undergo isolation from the reaction mixtures, or further

A. Bilyk et al. / Progress in Organic Coatings 62 (2008) 40–48

purification, prior to their dilution and application to oxidized film surfaces. 2.6. Surface activation and characterisation Low-density polyethylene (LDPE) film without additives was supplied by Goodfellows. Prior to surface modification, the films were washed with isopropanol and distilled water, and then airdried. One surface of each of the LDPE films (100 mm × 50 mm) was oxidized in air using a corona discharge (Tantec Electrical Surface Treatment System; Model HV 05-1), with the following treatment parameters: treatment speed: 0.36 m/min; substrate–electrode distance: 5 mm; power output: 100 W over a discharge length of 15 cm. The surface energy of the film before and after treatment was determined using dyne pens. An energy increase from 30 dynes/cm to 60 dynes/cm was achieved using the abovementioned treatment parameters. No attempt was made to further optimise the conditions. XPS analysis was conducted using a Kratos Axis HS spectrometer equipped with a monochromatic A1 K␣ X-ray source. The surface charge was compensated by the equipped charge neutraliser. The surface area analysed was approximately 1 mm2 and the spectra were collected at 0◦ emission (normal to the surface). The power applied for the analysis was 120 w. The hydrocarbon peak at 285 eV served as an internal reference. Chemical compositions expressed as atomic percentage were calculated from the survey spectra collected over 20 min at 0.5 eV step. Values stated in this work represent the average values from measurements on duplicate films. Tapping Mode AFM (Nanoscope Multimode AFM; Digital Instruments) was used to characterise surfaces, using super sharp etched silicon cantilevers with a radius of curvature of 2 nm and a resonant frequency of 320 kHz. In order to ensure the reproducibility of results scans were acquired on two to three areas of the sample surface. Parameters such as scan speed and set point ratio (ratio of the amplitude of the cantilever oscillation to the imaging set point to the amplitude of its oscillation in free space) were optimised to acquire scans, with the set point ratio being the same for all scans. The images presented in this report have a dimension of 5 ␮m × 5 ␮m, and were produced at a scan rate of 0.5 Hz. 2.7. Application of surface coating The PEI polymers containing grafted AQ (PEI-g-AQ) were applied to the surface of six surface-activated LDPE film replicates. Each of the solutions containing the PEI-g-AQ reaction products was diluted with methoxyethanol (0.5%, w/w) to form the “carrier solution” prior to surface coating. The PEI-g-AQ polymers were then applied to one side of the surface-activated LDPE films by immersion of the film in the carrier solution for 5 s. Following immersion, the samples were left to air-dry for at least 1 h in the dark at room temperature, and were then stored in the dark in aluminium foil to avoid premature activation.

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2.8. Photoreduction analysis Each film was placed in an air-tight quartz cuvette and equilibrated in nitrogen for ∼4 h prior to exposing to the UV-A light source (Crompton Blacklights; 2 W × 20 W) emitting UV radiation with a maximum intensity at about 350 nm. UV–vis absorption spectra were measured using a Shimadzu UV–vis PharmaSpec UV-1700 spectrophotometer or a Varian Cary 3E UV–vis spectrophotometer. The relative photoreduction efficiencies were estimated by monitoring the increase in absorbance by the reduced AQ species at 280 nm, and the decline in absorbance by the AQ compound at 260 nm, on exposure to the UV light. After irradiation, samples were exposed to air and the re-oxidation of the reduced species was monitored over a 90-min period. 2.9. Hydrogen peroxide production analysis A Merck Reflectoquant and the associated Merck hydrogen peroxide test strips (552), having a detection range of 0.2–20 ppm, were used to test for the presence of hydrogen peroxide after re-oxidation of the UV-irradiated films, following the method of analysis specified by the manufacture. This method was calibrated using a series of standard aqueous hydrogen peroxide solutions prepared from a 3% hydrogen peroxide solution (Sigma–Aldrich). The concentration of the 3% hydrogen peroxide solution was confirmed by titration against a standardised potassium permanganate solution. 3. Results and discussion 3.1. Analysis of grafted polymers used for coatings The modified PEI polymers prepared in this work will be referred to generically as PEI-g#-GLYAQ, where the italicized number refers to the level (% w/w) of the AQ component of the GLYAQ incorporated in the modified PEI. Analysis of the Q-13 C NMR spectra for both modified PEI polymers revealed two common chemical environments/shifts at approximately 68 ppm and 70 ppm. These resonances occur at chemical shifts that are very similar to that in the Q-13 C NMR spectrum of the model compound (2) and which is assigned to the methine (2 ) hydroxyl carbon that is formed upon ring opening of the glycidyl moiety of GLYAQ (1) via reaction with a primary amine. Therefore, these two distinguishable shifts in the Q-13 C NMR spectra of the PEI-g-AQ polymers are attributed to the methine (2 ) hydroxyl carbon that is formed upon ring opening of the glycidyl moiety via reaction with the primary or secondary amino groups of PEI. Since the grafting of GLYAQ to PEI was conducted under essentially the same reaction conditions as for the synthesis of the model compound (2), the resonances near 68 ppm and 70 ppm in the Q-13 C NMR spectra of the PEI-g-GLYAQ polymers are unlikely to be due to the opening of the glycidyl ring resulting from reaction with either the solvent, or with traces of moisture in the solvent. Furthermore, broad resonances associated with the AQ fragment were observed in the 1 H NMR spectra of the PEI-g-GLYAQ polymers, with no evidence of the

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sharp peaks expected for unbound AQ, providing qualitative evidence of the expected restricted molecular motion arising from covalent grafting of GLYAQ to PEI. A shoulder on the neighbouring resonance due to residual un-deuterated chloroform overlaps with the resonances at 68 ppm and 70 ppm preventing the quantitative determination of the amounts of covalently attached AQ in the PEI-g-GLYAQ polymers. Nevertheless, the results from analysis of the Q-13 C NMR spectra of the modified PEI polymers not only indicate the presence of GLYAQ grafted to PEI, but also provide semi-quantitative evidence that the level of grafted GLYAQ in PEI-g8.7-GLYAQ was close to the target level. Although the targeted level of GLYAQ substitution for PEI-g4.9-GLYAQ proved too low to analyse the resulting Q-13 C NMR spectrum with any confidence, TLC analysis of the PEI-g-GLYAQ polymers indicated the absence of residual unreacted GLYAQ compounds in the reaction mixtures, implying that covalent attachment of GLYAQ to the PEI was close to quantitative for both polymers. The origin of the resonances observed in the Q-13 C NMR spectrum of PEI-g4.9-GLYAQ at around 160 ppm has not been identified at this stage, but they might possibly be associated with the carbonyl groups of carbamates that are known to form on reaction between the amino groups PEI with atmospheric carbon dioxide [19,20], although resonances at similar chemical shifts were not detected in the Q-13 C NMR spectrum of PEI-g8.7-GLYAQ. On the basis of the spectroscopic and chromatographic evidence described above, it is concluded that GLYAQ was successfully covalently attached to the amino groups of PEI, and in the following discussion the actual amount of the AQ derivatives in the PEI-g-GLYAQ polymers will be considered to be effectively the target levels used in the syntheses. A schematic structure of the PEI polymer modified with grafted GLYAQ is shown in Fig. 1. 3.2. Analysis of film surface and coating The chemical compositions (relative elemental ratios) of the LDPE film surfaces before and after coating with PEI-g-GLYAQ were determined by XPS analysis, and the results obtained are summarised in Table 1 (the XPS survey scans are available as supplementary information). From these results it can be seen that the oxygen content on the surface increases after corona treatment, confirming oxidative functionalisation of the surface of the LDPE films. Subsequent treatment of the films with the solutions containing PEI-g-GLYAQ results in the presence of both nitrogen and sulfur on the surface, together with

Fig. 1. Schematic structure of PEI-g-GLYAQ showing GLYAQ grafted to a primary amine of PEI.

a concomitant decrease in the relative oxygen content, which is qualitatively consistent with the presence of PEI-g-GLYAQ coatings on the surface of the LDPE films. Although the nitrogen to sulfur (N/S) ratios calculated for the coated films do not appear to correlate well with the level of AQ in the PEI-g-GLYAQ polymers, it should be noted that the experimentally determined elemental ratios for the surface of a treated film are often not in quantitative agreement with the theoretical values. This is usually attributed [21–23] to contributions to the apparent surface composition arising from absorption of hydrocarbon contaminants on exposure of the treated films to air, and the preferential arrangement of the attached polymer’s hydrophobic components towards the outer surface of the coating (to reduce surface energy). AFM images of the LDPE film surfaces before and after application of the PEI-g-GLYAQ polymer coatings are shown in Fig. 2. The fine fibrillar structure depicted in the AFM image of the untreated, virgin LDPE surface is characteristic of the biaxial orientation that can occur on processing polyolefin materials. Following corona treatment, spherical mounds appear at the surface, which tend to blanket the fine underlying fibrillar structure at the surface of the film. The globular spherical mounds are composed of what is referred to as “low molecular weight oxidized material” (LMWOM), and their formation has been previously reported for polyolefins following corona treatment [24]. As shown in Fig. 2, after application of the PEI-g-GLYAQ solutions to the corona-treated surfaces, a significant decrease in the surface roughness compared with that of the initial corona-treated surface was observed. This result confirms that the PEI-g-GLYAQ polymers coated the corona-treated surface. Analysis of the AFM images indicates there was not an

Table 1 Chemical composition of film surface as determined by XPS Sample

C (%)

O (%)

N (%)

S (%)

Si (%)

N/Ca

N/Sb

LDPE control (no treatment) Corona-treated LDPE LDPE/PEI-g4.9-GLYAQ LDPE/PEI-g8.7-GLYAQ

99.35 82.46 72.96 74.23

0.50 17.11 4.46 5.80

0.05 0.42 22.37 19.58

– 0.01 0.21 0.39

0.10 – – –

0.001 0.005 0.307 0.264

– – 106.5 50.2

a b

Ratio of nitrogen to carbon. Ratio of nitrogen to sulfur.

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Fig. 2. AFM images of the film surfaces before and after corona-treatment and coating: (a) untreated control; (b) corona treated; (c) corona treated + 0.5% PEI-g4.9GLYAQ; (d) corona treated + 0.5% PEI-g8.7-GLYAQ.

appreciable difference in the surface roughness of the coatings obtained using the two types of PEI-g-GLYAQ polymers prepared in this work, with the RMS roughness for the PEI-g4.8GLYAQ and PEI-g8.7-GLYAQ coatings being 1.2 ± 0.1 nm and 1.9 ± 0.1 nm, respectively (average RMS values for five discrete areas of the AFM profiles of the coated films). The absorption spectra of the films after applying the PEI-g-GLYAQ coatings are shown in Fig. 3. The thickness of the PEI-g4.9-GLYAQ and PEI-g8.7-GLYAQ coatings were estimated to be 200 ± 60 nm and 90 ± 20 nm, respectively, by using the absorbance of the AQ component at 260 nm (ε = 3.5 × 104 M−1 cm−1 ) after subtracting the absorbance of the untreated LDPE film, and the levels of anthraquinone in each of the PEI-g-GLYAQ polymers. The PEI-g-GLYAQ coating is likely to be inter-diffused to some extent with the oxidized surface layer of the LDPE, leading to some dilution of the effective concentration of the AQ chromophore at the coated

surface. Therefore, these coating thicknesses should be considered as “apparent” coating thicknesses rather than necessarily the thickness of a completely discrete layer of PEI-g-GLYAQ. Furthermore, from the above-mentioned RMS roughness values, it can be concluded that the higher absorbance of the PEIg4.8-GLYAQ coated LDPE films compared with that of the PEI-g8.7-GLYAQ coated LDPE films is not the result of greater surface area/roughness of the PEI-g4.8-GLYAQ coating. Based on the MW of the PEI repeat unit (44 g/mol), the degree of substitution of PEI amino groups with the GLYAQ moiety is estimated to be around 1% and 2% for PEI-g4.9-GLYAQ and PEI-g8.7-GLYAQ, respectively. Despite this relatively low level of substitution, these amino groups have been substituted with the much more bulky GLYAQ group whose presence is likely to significantly disrupt the strength of binding at the interface between the PEI and the PE. Therefore, it seems reasonable to attribute the markedly thinner coating of PEI-g8.7-GLYAQ compared with PEI-g4.9-GLYAQ as being due to a substantial reduction in binding strength of PEI-g-GLYAQ to the coronatreated LDPE caused by the increased steric effects associated with the increased content of the bulky GLYAQ in the polymer. 3.3. Photoreduction

Fig. 3. Absorption spectra of LDPE films (in triplicate) coated with PEI-g4.9GLYAQ (—) and PEI-g8.7-GLYAQ (– – –).

Evaluation of the optimal conditions for photoreduction was conducted for the LDPE films coated with PEI-g-GLYAQ. The photoreduced form of the AQ components reacts rapidly with oxygen, so for the purposes of initial evaluation of photoreduction performance, the samples were irradiated in an atmosphere of nitrogen in order to limit the photoreduction to a single cycle. The samples were contained in nitrogen-purged cuvettes and were irradiated under UV lamps until the absorbance associated with the photoreduced species at around 280 nm reached

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Fig. 4. (a) LDPE film coated with PEI-g4.9-GLYAQ irradiated in N2 . Irradiation time: (a) before irradiation, (b) 1 min, (c) 2 min, (d) 3 min, (e) 6 min. and (f) 8 min. (b) LDPE film coated with PEI-g4.9-GLYAQ exposed to air after irradiating in N2 : before irradiation (—); after irradiating for 8 min (– · · –); after re-oxidizing in air for 20 min (– – –).

a maximum. The changes in the spectra of the samples during irradiation are shown in Fig. 4(a) and Fig. 5(a). After optimal photoreduction appeared to have been reached, the samples were then exposed to air in the dark, and the spectral changes on re-oxidation are shown in Figs. 4(b) and 5(b). As seen from the spectra in Figs. 4(a) and 5(a), the absorbance at 280 nm initially increases with UV irradiation. The absorbance then ceases to display any further measurable increase, indicating all of the photoreducible AQ moieties have been reduced, and this is considered to be the optimal UV dose for the given coating formulation. The optimal exposure time for the coated films was found to be between 5 min and 8 min for the conditions used in this work, where optimal exposure at a given AQ concentration will depend on film thickness. It can also be seen from the spectra in Figs. 4(b) and 5(b) that the absorbance at 280 nm decreases on exposure of the films to air in the dark, with the absorbance at around 260 nm due to the AQ being recovered almost quantitatively within about 20 min. It is assumed that this oxidative conversion of the photoreduced species back to AQ is accompanied by a concomitant production of hydrogen peroxide. In order to evaluate the photoreduction behaviour of the coating under more realistic end-use conditions, an LDPE film coated with PEI-g4.9-GLYAQ was irradiated while exposed to air. The change in the absorption spectrum of the sample during irradiation is shown in Fig. 6. Clearly, the increase in absorbance at 280 nm, associated with the photoreduced species, that was

Fig. 5. (a) LDPE film coated with PEI-g8.7-GLYAQ irradiated in N2 . Irradiation time: (a) before irradiation, (b) 1 min, (c) 3 min, and (d) 5 min. (b) LDPE film coated with PEI-g8.7-GLYAQ exposed to air after irradiating in N2 : before irradiation (—); after irradiating for 5 min (– · · –); after re-oxidizing in air for 20 min (– – –).

observed for the samples irradiated under nitrogen is not evident when the irradiation is conducted in the presence of air. The photoreduction/re-oxidation cycle occurs so rapidly in the presence of air that accumulation of the photoreduced species is unlikely, so it is not surprising that this species is not detected

Fig. 6. LDPE film coated with PEI-g4.9-GLYAQ exposed to air after irradiating in air: before irradiation (—); after irradiating for 8 min (– · · –); after re-oxidizing in air for 20 min (– – –).

A. Bilyk et al. / Progress in Organic Coatings 62 (2008) 40–48 Table 2 Hydrogen peroxide extracted from PEI-g-GLYAQ coatings on LDPE films after UV irradiation in air or vacuum Coating

PEI-g4.9-GLYAQ PEI-g8.7-GLYAQ

H2 O2 yield in air

H2 O2 yield in vacuum

ppm

%

ppm

%

0.7 0.5

52 46

2.8 2.9

208 267

under these conditions. The slight change to the shape of the AQ spectrum at wavelengths shorter than about 260 nm suggests that a product/s other than AQ might also be produced under these conditions. 3.4. Hydrogen peroxide production Replicates of LDPE films (50 mm × 50 mm) coated with either PEI-g4.9-GLYAQ or PEI-g8.7-GLYAQ were irradiated either in the absence of air by first vacuum packing the films into polyethylene bags, or in the presence of air. The duration of UV irradiation for both sets of samples was 8 min, and subsequent exposure to air in the dark was for 1 h. The films were then immersed in distilled water (3 ml) for 1 h, with no attempt made in this work to optimise the duration of immersion. The films were removed from the water, and the amount of hydrogen peroxide extracted into the water was tested. The levels of hydrogen peroxide detected for each coated film are summarised in Table 2.

47

The yields of hydrogen peroxide shown in Table 2 are the amounts of hydrogen peroxide detected as a ratio of the amount produced theoretically from a single photoreduction/reoxidation cycle, where it is assumed that every AQ moiety is photoreduced and that every photoreduced AQ moiety reacts with a molecule of oxygen to produce one molecule of hydrogen peroxide. Calculation of the theoretical values also requires the use of the estimated coating thicknesses mentioned above. For the coated films irradiated in air, the experimentally determined amounts of hydrogen peroxide are smaller than those predicted theoretically, and this is attributed to the efficiency of photoreduction of the AQ moiety being less than 100% due to the absence of labile hydrogen atoms around some of the AQ moieties. It is also possible that the spectral shape change that occurs on irradiation in air (see Fig. 6) is due to the occurrence of other reactions that compete with the desired photoreduction/reoxidation reactions when the coated film is irradiated in air. The slightly greater surface roughness/area of the PEI-g8.7GLYAQ coating might be expected to result in consistently higher efficiencies of hydrogen peroxide generation than for the PEI-g4.8-GLYAQ coating, irrespective of exposure of the films to air or vacuum. However, this is not borne out by the data in Table 2, suggesting that the magnitude of the difference in surface roughness of the two coatings is probably too small to have any significant bearing on the measured efficiencies. At present it is not clear why the amounts of hydrogen peroxide produced by coated films irradiated in vacuum, which presumably undergo only a single redox cycle, are substantially larger than that produced by coated films irradiated in air, and

Scheme 1.

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Fig. 7. Absorption spectra of LDPE films (in triplicate) coated with PEI-g8.7GLYAQ before rinsing (—) and after rinsing (– – –).

also the amounts predicted theoretically. One possible explanation is that, as illustrated in Scheme 1, the re-oxidation reaction involving reaction of the photoreduced AQ with oxygen proceeds via formation of two hydroperoxy radicals which then, subsequently, extract hydrogen atoms from the polymer chain to result in the formation of two molecules of hydrogen peroxide for each oxygen molecule consumed in the re-oxidation step. 3.5. Effect of rinsing on coating thickness The strength of the binding of the PEI-g-GLYAQ coating to the treated LDPE film surface was investigated by rinsing coated films four times with distilled water immediately after immersion in the carrier solution. Previous studies [24] have shown that the globular mounds of LMWOM are loosely attached and can be easily modified or removed by washing the surface with a solvent such as isopropanol or water, and so it is expected that rinsing should be sufficient to remove most of any PEI-g-GLYAQ that might be unbound to the treated LDPE surface. The absorption spectra of the films after applying the PEIg8.7-GLYAQ polymer coatings, followed by rinsing, are shown in Fig. 7. The thicknesses of the coating, calculated using the absorbance of the AQ component at 260 nm after subtracting the absorbance of the untreated LDPE film, was found to decrease to around 60 ± 10 nm (compared with 90 ± 20 nm prior to rinsing), indicating that unbound PEI-g8.7-AQ is removed from the surface during rinsing. Preliminary results for a PEI polymer containing 15% w/w AQ moiety (29% w/w grafted GLYAQ) suggest that increasing the level of grafted AQ moiety beyond around 9% w/w leads to a further substantial decrease in the level of binding of the PEI-g-GLYAQ to the corona-treated LDPE surface, and this phenomenon is attributed mainly to the weakening of the binding strength caused by the steric hindrance associated with the bulky GLYAQ substituents grafted onto the PEI. 4. Conclusions This work has demonstrated that the powerful microbial control agent hydrogen peroxide can be produced at the surface of a polyethylene film by applying a nano-scale polymeric coat-

ing comprising anthraquinone moieties covalently bound to the amine groups of PEI. Further work is required to determine whether the amount of hydrogen peroxide produced at the film surface is sufficient to be effective for the inactivation of microorganisms such as vegetative cells, spores, fungi, and viruses. It was found that the strength of binding of the PEI-g-GLYAQ polymeric coating to the surface of the oxidized polyethylene decreases markedly as the level of grafted anthraquinone increases, which is attributed to weakening of the binding strength caused by the steric hindrance associated with the bulky GLYAQ substituents grafted onto the PEI. Modification of the surface of substrates other than thin polymer films studied in this work is possible, in principle, and could find application in the control of microbial proliferation on other polymeric surfaces relevant to the food and health industries. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.porgcoat.2007.09.011. References [1] J.H. Han (Ed.), Innovations in Food Packaging, Elsevier, 2005 (Chapter 6). [2] P. Suppakul, J. Miltz, K. Sonneveld, S.W. Bigger, J. Food Sci. 68 (2003) 408–420. [3] A.L. Brody, E.R. Strupinsky, L.R. Kline, Active packaging for food applications, Technomic (Chapter 10). [4] T.L. Vigo, Polym. News 21 (1996) 82–86. [5] P. Appendini, J.H. Hotchkiss, J. Appl. Polym. Sci. 81 (2001) 609–616. [6] J. Vartiainen, M. R¨att¨o, U. Tapper, S. Paulussen, E. Hume, Polym. Bull. 54 (2005) 343–352. [7] J. Vartiainen, M. R¨att¨o, S. Paulussen, Packag. Technol. Sci. 18 (2005) 243–251. [8] G.W. Halek, A. Garg, J. Food Saf. 9 (1989) 215–222. [9] X. Qu, A. Wirs´en, B. Olander, A.-C. Albertsson, Polym. Bull. 46 (2001) 223–229. [10] J. Sherrill, S. Michielsen, I. Stojiljkovic, J. Polym. Sci. Polym. Chem. A 41 (2003) 41–47. [11] J. Bozja, J. Sherrill, S. Michielsen, I. Stojiljkovic, J. Polym. Sci. Polym. Chem. A 41 (2003) 2297–2303. [12] M.D.I.A. Ansari, A.K. Datta, Trans. IChemE 81 (2003) 57–65. [13] H. G¨orner, Photochem. Photobiol. 77 (2003) 171–179. [14] V.P. Foyle, Y. Takahashi, J.E. Guillet, J. Polym, Sci. A: Polym Chem. 30 (1992) 257–269. [15] C.F. Albert, M.L. Rooney, US Patent 6,123,901. [16] Bilyk, S. Li, W.D. Yang, P.M. Hoobin, I.J. Russell, W.S. Gutowski, US Patent 6,800,331. [17] M.L. Rooney, M.A. Horsham, US Patent 6,601,732. [18] A.M. Aquino, C.J. Abelt, K.L. Berger, C.M. Darragh, S.E. Kelley, M.V. Cossette, J. Am. Chem. Soc. 112 (1990) 5819–5824. [19] D.B. Dell’amico, F. Calderazzo, L. Labella, F. Marchetti, G. Pampaloni, Chem. Rev. 103 (2003) 3857–3898. [20] S. Satyapal, T. Filburn, J. Trela, J. Strange, Energy Fuels 15 (2001) 250–255. [21] T.L. Barr, S. Seal, J. Vac. Sci. Technol. A13 (1995) 1239. [22] J.D. Andrade (Ed.), Polymer Surface Dynamics, Plenum Press, New York, 1988. [23] F. Garbassi, M. Morra, E. Occhiello, Polymer Surfaces—From Physics to Technology, Wiley, Chichester, 1994 (Chapter 2). [24] V. Jones, M. Strobel, M.J. Prokosch, Plasma Process. Polym. 2 (2005) 547–553.